The flagellated protozoan parasite Trypanosoma brucei is responsible for African trypanosomiasis, which causes widespread mortality and morbidity of humans and livestock in sub-Saharan Africa. Sleeping sickness is fatal if untreated, yet no vaccine exists and current treatments are old, toxic and difficult to administer. Thus, there is a pressing need for research to better understand these parasites and facilitate development of new therapeutic interventions. T. brucei alternates between mammalian and tsetse fly hosts and must integrate signals from the external environment for survival, development and pathogenesis in both hosts. Parasite surface proteins protect against host immune defenses, allow acquisition of nutrients and provide sensory and signaling functions important for host-parasite interactions and pathogenesis. Cell surface proteins are thus critical for the parasitic life-style of T. brucei and make attractive targets for therapeutic intervention in sleeping sickness. To date, no systematic study of the parasite surface proteome has been conducted and key surface-exposed proteins remain mostly unknown. This represents a major gap in our understanding of trypanosome biology and the host-parasite interface. By combining affinity purification of cell surface proteins with state-of-the-art proteomics and a pioneering method for isolating intact flagella, we are now able to define the surface proteome of T. brucei, including surface sub-domains that are specialized for signaling and host-parasite interaction. Our goals are to capitalize on our proteomics approach to define the complete protein repertoire of the T. brucei cell surface from insect and mammalian bloodstream form parasites. From these data and our flagellum proteome, we will identify host-specific surface proteins, which will be tested for an impact on infection of the mammalian host. While our focus is on the host-parasite interface of T. brucei, we expect our results to be of wide interest for the community studying fundamental biology and pathogenesis of parasitic protozoa.